Hemophilias A and B are caused by heterogeneous mutations in the genes on chromosome X that encode for the coagulation factor VIII (F8) (Kemball-Cook G, Tuddenham E G, Nucleic Acids Res., 25:128-132 (1997)) and coagulation factor IX (F9) (Giannelli F, Green P M, Sommer S S, et al., Nucleic Acids Res., 26:265-268 (1998)), respectively. There is a 25% chance for a pregnant hemophilia carrier to have an affected male fetus in each pregnancy. Prenatal diagnosis is an important aspect of reproductive choices for women in families with hemophilia (Lee C A, Chi C, Pavord S R, et al., Haemophilia., 12:301-336 (2006)). In addition, it is also beneficial for appropriate obstetric management during labor and delivery as prolonged labor, invasive monitoring techniques and instrumental deliveries should be avoided in affected fetuses to minimize potential fetal and neonatal hemorrhagic complications (Lee C A, Chi C, Pavord S R, et al., Haemophilia., 12:301-336 (2006)). Therefore, the development of a noninvasive prenatal diagnostic approach for hemophilia is beneficial to both obstetricians and hemophilia families.
Current prenatal diagnostic methods for sex-linked diseases are typically invasive and pose a risk to the fetus. The discovery of cell-free fetal DNA in maternal plasma has offered new opportunities for noninvasive prenatal diagnosis (Lo Y M D et al., Lancet., 350:485-487 (1997); Lo Y M D, Chiu R W K, Nat Rev Genet., 8:71-77 (2007)). A number of promising clinical applications have been developed based on the detection of paternally inherited genetic traits in maternal plasma. For example, the noninvasive detection of fetal sex and RHD status are useful for the clinical management of sex-linked diseases and RhD incompatibility (Bustamante-Aragones A et al., Haemophilia., 14:593-598 (2008); Finning K et al., BMJ., 336:816-818 (2008)). For monogenic diseases such as achondroplasia and β-thalassemia, the detection of the presence or absence of paternally inherited mutations in maternal plasma would allow one to diagnose autosomal dominant diseases or exclude autosomal recessive diseases of the fetuses, respectively (Saito H et al., Lancet., 356:1170 (2000); Chiu R W K et al., Lancet., 360:998-1000 (2002); Ding C et al., Proc Natl Acad Sci USA., 101:10762-10767 (2004)).
Despite the rapid development of the field, it has remained difficult to detect fetal alleles that are inherited from mothers who are carriers for the mutations. The difficulty is caused by the coexistence of fetal and maternal DNA in maternal plasma, and the maternally inherited fetal allele is indistinguishable from the background maternal DNA (Lo Y M D, Chiu R W K, Nat Rev Genet., 8:71-77 (2007)).
Therefore, it is desirable to provide accurate and efficient methods for determining whether a male fetus has inherited an X-linked mutation.